Nucleic acids encoding lettuce big-vein viral proteins and utilization thereof

ABSTRACT

The coat protein of lettuce big-vein virus (LBVV) was purified from highly purified LBVV, and its partial amino acid sequences were determined. An RNA encoding the coat protein of LBVV was cloned by polymerase chain reaction using primers designed based on the determined amino acid sequences information, and its primary structure was elucidated. Moreover, the present inventors succeeded not only in isolating RNA molecules of a plurality of LBVV-encoded proteins, including LBVV polymerase, by carrying out 3′RACE and 5′RACE using primers designed based on the resulting sequence information, but also in determining their primary structure. It was found that the use of these made it possible to produce lettuce resistant to LBVV and to diagnose infections with LBVV.

TECHNICAL FIELD

The present invention relates to nucleic acids encoding lettuce big-veinviral proteins, proteins encoded by the nucleic acids, and theirproduction and use.

BACKGROUND ART

Lettuce big-vein virus (LBVV) is a virus belonging to Varicosavirus, iscomposed of two RNAs (7.0 kb and 6.5 kb RNA), and retains a coat proteinof 48 kDa. LBVV is a soil-borne virus that is spread in the soil byOlpidum brassicae, and occurs in the United States, Australia, NewZealand, Japan and Europe. Since this virus infects lettuce andremarkably lowers its quality and yield, it is a serious problem inlettuce production.

Unfortunately, there has not yet been reported the existence of a genethat makes lettuce resistant to this virus. Although several cultivarssuch as Entree, Sea Green and Pacific are commercially available asLBVV-resistant cultivars, their resistance is low. Thus, there has notyet been found a radical solution to disease damage caused by LBVV.

Elucidation of the virus genetic information is an important step inpreventing disease damage caused by the virus. However, isolation andpurification of LBVV are extremely difficult for reasons such as theinstability of the viral particles, tendency for viral particles toreadily aggregate with each other, and extremely low concentration ofthe virus in plants. Although, so far, two successful examples ofpurification of the virus have been reported (S. Kuwata et al., (1983),Annals of the Phytopathological Society of Japan, 49, 246-251; and, H.J. Vetten et al. (1987), Journal of Phytopathology, 120, 53-59), thereproducibility is low and the purified amounts are extremely low.Consequently, as to LBVV, no genetic information has been elucidated atall.

DISCLOSURE OF THE INVENTION

The present invention has been achieved in consideration of the abovecircumstances, and objectives of the present invention are to isolatelettuce big-vein viral (LBVV) proteins and nucleic acids that encode theproteins and to elucidate the structure thereof. In addition, anotherobjective of the present invention is to endow lettuce with resistanceto LBVV through expression of the nucleic acid or its antisense nucleicacid in lettuce. Moreover, still another objective of the presentinvention is to provide a method of diagnosing infection with LBVV bydetecting the nucleic acid or a protein encoded by the nucleic acid.

LBVV is an RNA virus, and it is likely that, if a DNA encoding a proteinof the virus or its antisense DNA is expressed in a plant, theproduction and function of LBVV proteins can be inhibited at thetranscription level or translation level (P. F. Tennant et al., (1994),Phytopathology, 84, 1359-1366; C. C. Huntley & T. C. Hall, (1993),Virology, 192, 290-297; D. C. Baulcombe, (1996), The Plant Cell, 8,1833-1844).

The present inventors focused on this idea, and isolated the genesencoding LBVV proteins in order to produce lettuce resistant to LBVV.

Specifically, the present inventors first obtained highly purified LBVV,and then applied this to SDS-polyacrylamide gel electrophoresis todetect the coat protein that constitutes the virus. The detected coatprotein was purified and then decomposed into peptides, followed bydetermination of the partial amino acid sequences of the peptides by theEdman's method. Moreover, an RNA encoding the coat protein of LBVV wascloned by polymerase chain reaction (PCR) using primers designed on thebasis of information from the determined amino acid sequences, followedby determination of its nucleotide sequence.

Next, in order to determine the gene that encodes the full-length coatprotein of LBVV, RNAs were prepared from purified virus and from leafinfected with the virus that had exhibited obvious symptoms ofinfection, and 3′RACE and 5′RACE were carried out using these RNAmolecules. As a result, the present inventors have succeeded not only inisolating the RNA molecule that encodes LBVV coat protein, but also indetermining its primary structure. Moreover, by genome walking method,the present inventors have succeeded in isolating RNA molecules encodingfour other non-structural proteins of LBVV and in determining theirprimary structures as well.

Similarly, the present inventors also succeeded in isolating an RNAmolecule that encodes a polymerase protein from highly purified LBVV.

The isolated RNA molecule or its antisense molecule is able to endowlettuce plants with resistance to LBVV by its expression, and thereby,it is possible to improve lettuce productivity. In addition, geneticdiagnosis of LBVV can also be carried out by designing and using aprimer specific to LBVV based on sequence information of the isolatedRNA molecules. Furthermore, the antisera that bind to LBVV proteins canbe produced based on the resulting sequence information, and these canbe used for serological diagnosis of LBVV.

The present invention was completed on the basis of the above findings,and provides LBVV proteins, nucleic acids encoding the proteins, andtheir production and use.

More specifically, the present invention provides the following:

(1) a nucleic acid encoding a protein of lettuce big-vein virus, saidnucleic acid selected from the group consisting of:

-   -   (a) a nucleic acid encoding a protein comprising the amino acid        sequence of any one of SEQ ID NOs: 2 through 6 and SEQ ID NO:        13; and    -   (b) the nucleic acid of (a) comprising the coding region of the        nucleotide sequence of SEQ ID NO: 1 or 12;

(2) the nucleic acid according to (1), wherein the nucleic acid is anRNA;

(3) the nucleic acid according to (1), wherein the nucleic acid is aDNA;

(4) a DNA encoding a sense RNA complementary to a complementary strandof the nucleic acid according to (2);

(5) a DNA encoding an antisense RNA complementary to the nucleic acidaccording to (2);

(6) a DNA encoding an RNA having ribozyme activity that specificallycleaves the nucleic acid according to (2);

(7) a vector comprising the nucleic acid according to (3);

(8) a transformed cell comprising the nucleic acid according to (3), orthe vector according to (7);

(9) a protein encoded by the nucleic acid according to (1);

(10) an antibody that binds to the protein of (9);

(11) a method of producing the protein according to (9) wherein saidmethod comprises the steps of:

-   -   (a) culturing the transformed cell of (8); and    -   (b) recovering an expressed protein from said transformed cell        or its culture supernatant;

(12) a vector comprising the DNA according to any one of (4) through(6);

(13) a transformed lettuce cell that comprises the nucleic acidaccording to (1), the DNA according to any one of (4) through (6), orthe vector according to (7) or (12);

(14) a transformed lettuce plant comprising the transformed lettuce cellaccording to (13);

(15) a transformed lettuce plant that is a progeny or a clone of thetransformed lettuce plant according to (14);

(16) a propagation material of the transformed lettuce plant accordingto (14) or (15); and

(17) a method of diagnosing infection caused by the lettuce big-veinvirus wherein said method comprises the step of:

-   -   detecting the nucleic acid of (1), or detecting the protein        of (9) in lettuce cells; in Olpidum brassicae, a fungal vector        of lettuce big-vein virus; or in soil comprising the fungal        vector.

The present invention provides LBVV proteins and nucleic acids encodingthe proteins. The nucleotide sequence of cDNA that encodes LBVV proteinsisolated by the present inventors and that is included in the presentinvention is shown in SEQ ID NO: 1, and the amino acid sequences of theproteins encoded by the cDNA are shown in SEQ ID NOs: 2 through 6. Theisolated cDNA is a 6078 nucleotides sequence and encodes five proteins.Protein 1 (coat protein: Example 1) has a translation initiation site atnucleotide 209 and encodes 397 amino acids (the isolated clone was named“LBVV-cp”/SEQ ID NO: 2); protein 2 (Example 3) has a translationinitiation site at nucleotide 1492 and encodes 333 amino acids (SEQ IDNO: 3); protein 3 (Example 3) has a translation initiation site atnucleotide 2616 and encodes 290 amino acids (SEQ ID NO: 4); protein 4(Example 3) has a translation initiation site at nucleotide 3842 andencodes 164 amino acids (SEQ ID NO: 5); and protein 5 (Example 3) has atranslation initiation site at nucleotide 4529 and encodes 368 aminoacids (SEQ ID NO: 6).

In addition, the nucleotide sequence of cDNA (Example 4) that encodes apolymerase of LBVV isolated by the present inventors and that is alsoincluded in the present invention is shown in SEQ ID NO: 12, and theamino acid sequence of the protein encoded by the cDNA is shown in SEQID NO: 13 (the isolated clone was named “LBVV-L”). The isolated cDNA isa 6793 nucleotides sequence, has a translation initiation site atnucleotide 337, and encodes 2040 amino acids.

Moreover, the present inventors have revealed that LBVV is anegative-strand RNA virus that contains more positive-strands in itsviral particles than usual.

This is the first example of demonstrating the genes and protein primarystructures of LBVV.

Nucleic acids encoding LBVV-cp protein (LBVV protein 1) LBVV proteins 2to 5, or LBVV-L protein according to the present invention include a DNAand an RNA. The DNA includes a cDNA and a chemically synthesized DNA,and the RNA includes a viral genomic RNA, mRNA, and synthetic RNA. Anucleic acid of the present invention can be prepared using conventionalmeans by a person with ordinary skill in the art. Specifically, a firststrand DNA can be synthesized by carrying out a reverse transcriptionreaction using, as a template, (1) an. RNA prepared by de-proteinizingpurified virus by a method such as the SDS-phenol method or (2) thetotal nucleic acids extracted from a virus-infected leaf by the CTABmethod and so on and using a primer designed from the sequence of anucleic acid of the present invention or a random primer. From the firststrand DNA prepared by this method, a second strand DNA can besynthesized according to the method of Gubler & Hoffman (U. Gubler andB. J. Hoffman, (1983), Gene 25, 263-269), and the nucleic acid of thepresent invention can be cloned in various commercially availableplasmids or phagemid vectors. Alternatively, a DNA encoding an RNA ofthe virus can be amplified by polymerase chain reaction using a primerdesigned from the sequence of a nucleic acid of the present inventionand using the first strand DNA as a template, and the nucleic acid ofthe present invention can be cloned by TA cloning using the pGEM®-Tvector and so on or by cloning in various commercially available plasmidvectors by adding a restriction enzyme site to the primer.

A nucleic acid of the present invention can also be used for thepreparation of recombinant protein and for the production of lettuceresistant to LBVV.

A recombinant protein is usually prepared by inserting a DNA encoding aprotein of the present invention into an appropriate expression vector,introducing the vector into an appropriate cell, culturing thetransformed cells, allowing the cells to express the recombinantprotein, and purifying the expressed protein. A recombinant protein canbe expressed as a fusion protein with other proteins so as to be easilypurified, for example, as a fusion protein with maltose binding proteinin Escherichia coli (New England Biolabs, USA, vector pMAL series), as afusion protein with glutathione-S-transferase (GST) (Amersham PharmaciaBiotech, vector pGEX series), or tagged with histidine (Novagen, pETseries). The host cell is not limited so long as the cell is suitablefor expressing the recombinant protein. It is possible to utilize yeastsor various animal, plant, or insect cells by change the expressionvector, besides the above described E. coli. A vector can be introducedinto a host cell by a variety of methods known to one skilled in theart. For example, a transformation method using calcium ions (Mandel, M.and Higa, A. (1970) Journal of Molecular Biology, 53, 158-162, Hanahan,D. (1983) Journal of Molecular Biology, 166, 557-580) can be used tointroduce a vector into E. coli. A recombinant protein expressed in hostcells can be purified and recovered from the host cells or the culturesupernatant thereof by known methods. When a recombinant protein isexpressed as a fusion protein with maltose binding protein or otherpartners, the recombinant protein can be easily purified by affinitychromatography.

The resulting protein can be used to prepare an antibody that binds tothe protein. For example, a polyclonal antibody can be prepared byimmunizing immune animals, such as rabbits, with a purified protein ofthe present invention or its portion, collecting blood after a certainperiod, and removing clots. A monoclonal antibody can be prepared byfusing myeloma cells with the antibody-forming cells of animalsimmunized with the above protein or its portion, isolating a monoclonalcell expressing a desired antibody (hybridoma), and recovering theantibody from the cell. The obtained antibody can be utilized to purifyor detect a protein of the present invention. The antibody of thepresent invention includes antiserum, polyclonal antibody, monoclonalantibody, and fragment thereof.

In the case of producing LBVV-resistant lettuce, a DNA that repressesthe production and function of LBVV proteins should be introduced intolettuce cells, and the resulting transformed lettuce cells should beregenerated.

A DNA encoding an RNA that hybridizes with either strand (sense strandor complementary strand) of an RNA encoding LBVV proteins can be used asthe DNA that represses the production and function of the LBVV proteins.

Examples of a DNA encoding an RNA that hybridizes with viral genomicsense strand and with mRNAs include a DNA that encodes an antisense RNAthat is complementary to the transcription product of a DNA encoding theprotein described in any one of SEQ ID NOs: 2 through 6 and SEQ ID NO:13 isolated by the present inventors (preferably, a DNA comprising acoding region of the nucleotide sequence described in SEQ ID NO: 1 orSEQ ID NO: 12). Herein, the term “complementary” also means notcompletely complementary so long as the production of LBVV proteins canbe effectively inhibited. The transcribed RNA has preferably 90% or morecomplementarity and most preferably 95% or more complementarity to theRNA encoding the target LBVV protein. Herein, the term “complementarity”refers to the percentage of the number of nucleotides that formcomplementary base pairs, to the total number of base pairs in a regionwhere the two sequences correspond to each other, in the case that thesequences are aligned so that the number of complementary base pairs maybe maximized.

The DNA that encodes a sense RNA complementary to a complementary strandof RNA encoding the protein described in any one of SEQ ID NOs: 2through 6 and SEQ ID NO: 13 isolated by the present inventors(preferably, an RNA comprising a coding region of the nucleotidesequence described in SEQ ID NO: 1 or SEQ ID NO: 12) can be used as aDNA encoding an RNA that hybridizes with a complementary strand of viralgenomic RNA. Herein, the term “complementary” also means not completelycomplementary so long as the production of LBVV proteins can beeffectively inhibited. The transcribed sense RNA has preferably 90% ormore complementarity and most preferably 95% or more complementarity tothe RNA (complementary strand) encoding the target LBVV protein.

In order to effectively inhibit the expression of the target gene, theabove described antisense and sense DNAs should be at least 15nucleotides long, more preferably at least 100 nucleotides long, andstill more preferably at least 500 nucleotides long. These DNAs aregenerally shorter than 5 kb, and preferably shorter than 2.5 kb.

In addition, it is likely that a DNA encoding a ribozyme that cleaves atleast one of the strands of an RNA that encodes LBVV proteins can beused as a DNA that represses the production of the LBVV proteins.

A ribozyme is an RNA molecule that has catalytic activities. There aremany ribozymes having various activities. Research on the ribozymes asRNA cleaving enzymes has enabled the design of a ribozyme thatsite-specifically cleaves RNA. While some ribozymes of the group Iintron type or the M1RNA contained in RNaseP consist of 400 nucleotidesor more, others belonging to the hammerhead type or the hairpin typehave an activity domain of about 40 nucleotides (Makoto Koizumi and EikoOhtsuka (1990) Tanpakushitsu Kakusan Kohso (Nucleic acid, Protein, andEnzyme) 35: 2191-2200).

The self-cleavage domain of a hammerhead type ribozyme cleaves at the 3′side of C15 of the sequence G13U14C15. Formation of a nucleotide pairbetween U14 and A at the ninth position is considered important for theribozyme activity. Furthermore, it has been shown that the cleavage alsooccurs when the nucleotide at the 15th position is A or U instead of C(M. Koizumi et al. (1988) FEBS Letters, 228: 228-230). If the substratebinding site of the ribozyme is designed to be complementary to the RNAsequences adjacent to the target site, one can create arestriction-enzyme-like RNA cleaving ribozyme which recognizes thesequence UC, UU, or UA within the target RNA (M. Koizumi et al. (1988)FEBS Letters, 239: 285; Makoto Koizumi and Eiko Ohtsuka (1990)Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme), 35:2191-2200; M. Koizumi et al. (1989) Nucleic Acids Research, 17:7059-7071). For example, in LBVV-cp gene, LBVV protein 2 to 5 genes, orLBVV-L gene (SEQ ID NO: 1 or 12), there are a plurality of sites thatcan be used as the ribozyme target.

The hairpin type ribozyme is also useful in the present invention. Ahairpin type ribozyme can be found, for example, in the minus strand ofthe satellite RNA of Tobacco ringspot virus (J. M. Buzayan, Nature 323:349-353 (1986)). This ribozyme has also been shown totarget-specifically cleave RNA (Y. Kikuchi and N. Sasaki (1992) NucleicAcids Research, 19: 6751-6775; Yo Kikuchi (1992) Kagaku To Seibutsu(Chemistry and Biology) 30: 112-118).

The ribozyme designed to cleave the target is fused with a promoter,such as the cauliflower mosaic virus 35S promoter, and with atranscription termination sequence, so that it will be transcribed inplant cells. However, if extra sequences have been added to the 5′ endor the 3′ end of the transcribed RNA, the ribozyme activity can be lost.In this case, one can place an additional trimming ribozyme, whichfunctions in cis to perform the trimming on the 5′ or the 3′ side of theribozyme portion, in order to precisely cut the ribozyme portion fromthe transcribed RNA containing the ribozyme (K. Taira et al. (1990)Protein Eng. 3: 733-738; A. M. Dzaianott and J. J. Bujarski (1989) Proc.Natl. Acad. Sci. USA 86: 4823-4827; C. A. Grosshands and R. T. Cech(1991) Nucleic Acids Research, 19: 3875-3880; K. Taira et al. (1991)Nucleic Acid Research, 19: 5125-5130). Multiple sites within the targetgene can be cleaved by arranging these structural units in tandem toachieve greater effects (N. Yuyama et al., Biochem. Biophys. Res.Commun. 186: 1271-1279 (1992)). By using such ribozymes, it is possibleto specifically cleave the transcription products of the target gene inthe present invention, thereby repressing the expression of the gene.

Vectors used for the transformation of lettuce cells are not limited solong as the vector can express an inserted DNA in the cells. Forexample, vectors comprising promoters for constitutive gene expressionin lettuce cells (e.g., cauliflower mosaic virus 35S promoter); andpromoters inducible by exogenous stimuli can be used. Examples ofsuitable vectors include pBI binary vector. The “lettuce cell” intowhich the vector is to be introduced includes various forms of lettucecells, such as cultured cell suspensions, protoplasts, leaf sections,and callus.

A vector can be introduced into lettuce cells by known methods, such asthe polyethylene glycol method, polycation method, electroporation,Agrobacterium mediated transfer, and particle bombardment. For example,the method described in the literature (S. Z. Pang et al., (1996), ThePlant Journal, 9, 899-909) is a preferable one.

Regeneration of a lettuce plant from transformed lettuce cells can becarried out by methods known to a person with ordinary skill in the artaccording to the type of lettuce cells. Examples of preferableregeneration methods are described in the literature (S. Enomoto, etal., (1990), Plant Cell Reports, 9, 6-9).

Once a transformed lettuce plant in which the DNA of the presentinvention is introduced into the genome is obtained, it is possible togain progenies from that plant by sexual propagation. Alternatively,plants can be mass-produced from propagation materials (for example,seeds, tubs, callus, protoplast, and so on) obtained from the plant, orprogenies or clones thereof. The present invention includes plant cellstransformed with the DNA of the present invention; plants includingthese cells; progenies and clones of the plants; and propagationmaterials of the plants and their progenies and clones.

In addition, the present invention provides a method of diagnosinginfection with LBVV. One embodiment of the diagnostic method of thepresent invention comprises detecting, using a primer or probe, a LBVVRNA or an RNA encoding the viral protein. Nucleic acid comprising atleast 15 nucleotides homologous or complementary to a DNA encoding theLBVV protein described in any one of SEQ ID NOs: 2 through 6 and 13 canbe used for the probe or primer. The nucleic acid is preferably nucleicacid that specifically hybridizes with a DNA encoding the LBVV proteindescribed in any one of SEQ ID NOs: 2 through 6 and 13.

The primer or probe may be labeled as necessary. Examples of labelsinclude a radioactive label.

In this diagnosis, for example, a test sample is prepared from lettucesuspected of being infected with lettuce big-vein virus, Olpidumharboring the virus, or soil containing the virus, and PCR using theabove primer or northern blotting using the above probe is carried outon the sample.

Another mode of the diagnostic method of the present invention is amethod characterized by detecting LBVV proteins using antibody. Antibodyused in this diagnosis can be prepared, for example, by synthesizingpeptide using the antigenic region estimated from the resulting aminoacid sequences (any of SEQ ID NOs: 2 through 6 and 13), by binding thepeptide to a carrier protein such as KLH or BSA, and by immunizingrabbits with this. In addition, the antibody can also be produced bytagging LBVV proteins with histidine using the QIAexpress Type IV Kit(QIAGEN), by expressing the tagged protein in E. coli, and by immunizingrabbits with the resulting protein. The antibody may be labeled asnecessary. Examples of labels include an enzyme label. In addition,instead of directly labeling the antibody itself, the antibody may belabeled via a substance such as protein A that binds to the antibody,followed by detection of the target protein.

In this diagnosis, a test sample is prepared from, for example, lettucesuspected of being infected with lettuce big-vein virus, Olpidumharboring the virus, or soil containing the virus, and then, ELISA orwestern blotting is carried out on the sample using the above antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is specifically illustrated below with referenceto the examples, but is not construed as being limited thereto.

EXAMPLE 1 Cloning of Coat Protein Gene of Lettuce Big-vein Virus (LBVV)

Contaminated soil was sampled from a lettuce (cultivar: Cisco) field inKagawa Prefecture, Japan that exhibited characteristic big-vein symptomsin 1997. The virus was maintained in resting spores in dry soil kept inthe laboratory. Cisco, a cultivar of lettuce, was used for viruspurification, and the virus was inoculated by regular transfer in soil.

Virus purification was carried out by modifying the method of Kuwata etal. (S. Kuwata, et al., (1983), Annals of the Phytopathological Societyof Japan, 49, 246-251). The First step of sedimenting virions bylow-speed centrifugation was omitted. The virus fraction was obtained bytreating with 1% Triton-X and 1% Briji-35, followed by C₂SO₄ densitygradient centrifugation. When the purified virus obtained by thispurification method was subjected to SDS-polyacrylamide electrophoresis,only a single 48 kDa band was detected. In addition, since only clustersof LBVV particles were observed by electron microscopy while otherimpurities were not observed, the resulting virus was presumed to haveconsiderably high purity.

After treatment of the purified virus with Proteinase K-SDS, extractionof viral nucleic acid was carried out by phenol/chloroform and ethanolprecipitation. The purified viral nucleic acid was used for synthesis offirst strand cDNA after denaturing with dimethylsulfoxide. Poly(A)+RNAwas isolated from the virus-infected lettuce leaf that exhibited obviousbig-vein symptoms by using the Dynabeads® mRNA DIRECT™ Kit (Dynal®).Synthesis of first strand cDNA was performed by carrying out a reversetranscription reaction with SUPERSCRIPT™ II Rnase H⁻ ReverseTranscriptase (Gibco BRL) using a random primer or an Oligo-dT-Bam HIprimer.

Determination of the internal amino acid sequences of LBVV coat proteinwas carried out in the manner described below. After purified LBVV wassubjected to 12.5% SDS-polyacrylamide electrophoresis, the polypeptideswere transferred to a nitrocellulose membrane and the band of interestwas cut out followed by carboxymethylation, and treatment with lysylendopeptidase. After the treatment, 38 peptide fragments of the LBVVcoat protein were obtained by reverse phase HPLC, and the amino acidsequences of several internal peptide fragments were determined.

5LB111 primer (GARWSITGGGAYGAYGARWSIAC/SEQ ID NO: 7) and 3LB171 primer(GCRTCDATRTARTCIACICCIGG/SEQ ID NO: 8) were designed based onESWDDESTIAMP (SEQ ID NO: 17) and NLEVPGVDYIDA (SEQ ID NO: 18) of theresulting amino acid sequences. PCR was carried out using these primersand Takara Taq (Takara), a 274 bp PCR product was obtained. Theresulting PCR product was cloned using pGEM®-T Easy Vector Systems(Promega) and its nucleotide sequence was determined.

In order to determine the full-length coat protein gene of LBVV, 3′RACEor 5′RACE were aimed using an RNA from the purified virus or aPoly(A)+RNA from the LBVV-infected leaf. In the case of 3′RACE, 891-bpPCR product was obtained using a Poly(A)+RNA from the LBVV-infectedleaf, and using Olido-dT-Bam HI primer and 5LB171 primer(AAYYTIGARGTICCIGGIGTIGA/SEQ ID NO: 9). In the case of 5′RACE, a 760-bpPCR product was obtained with the 5′RACE System for Rapid Amplificationof cDNA Ends, Version 2.0 (Gibco BRL) using an RNA from purified virusor a Poly(A)+RNA from the LBVV-infected leaf, and using 3LB5R4 primer(GTTTTTGACCCTGATAG/SEQ ID NO: 10) and 3LB5R5 primer(GTCGACTCTAGACACTTGTTGTTTGTCGTG/SEQ ID NO: 11). The resulting PCRproducts were cloned using pGEM®-T Easy Vector Systems (Promega), andthe nucleotide sequences were determined for at least six clones ormore. In addition, the 500- to 700-bp PCR products from the region inthe vicinity of the coat protein gene were recloned using mutuallyoverlapping virus-specific primers. At least three clones were sequencedfrom each region, and the nucleotide sequence of coat protein gene wasconfirmed.

A 1425 nucleotides sequence was determined using the above method. Thisgene had a translation initiation site at nucleotide 209, and encoded397 amino acids (see SEQ ID NO: 1).

EXAMPLE 2 Production of Transformed Lettuce

(1) Sterilization and Culturing of Lettuce Seeds

Lettuce seeds were immersed for several seconds in 70% ethanol followedby treating for 15 minutes in a sterilization solution (10% sodiumhypochlorite, 0.05% Tween-20). Next, the seeds were rinsed withsterilized water, seeded on Hyponex agar medium (prepared by dissolving3.0 g of Hyponex powder, 10.0 g of sucrose and 8.0 g of agar in oneliter of distilled water and then adjusting the pH to 5.8 with 1 N NaOH)in a plant box, and grown for about 2 weeks under the light condition at25 to 28° C. until the true leaf reached about 5 cm.

(2) Culturing and Inoculation of Agrobacterium

Agrobacterium was inoculated into YEB liquid medium (prepared bydissolving 1.0 g of yeast extract, 5.0 g of beef extract, 5.0 g ofpeptone, 5.0 g of sucrose and 0.5 g of MgSO₄ 7H₂O in one liter ofdistilled water and then adjusting the pH to 7.0 with 1 N NaOH)comprising 250 μg/ml streptomycin, 5 μg/ml rifampicin and 50 μg/mlkanamycin, and then cultured with shaking overnight at 28° C. TheAgrobacterium culture liquid was then sub-cultured to fresh YEB medium(comprising the above-mentioned antibiotics) and additionally culturedwith shaking for one day at 28° C.

The young lettuce plants in which the true leaf had grown to about 5 cmwere transferred to plastic Petri dishes, the true leaf was cut intopieces measuring about 5 mm, and the pieces of the leafs were immersedfor 1 minute in Agrobacterium culture liquid diluted ten-fold. Next, thepieces were arranged on Murashige & Skoog medium (MS medium) (pH 5.8)comprising 3% sucrose, 0.5 ppm benzyladenine phosphate (BAP), 0.1 ppmnaphthalene acetic acid (NAA) and 0.8% agar at 15 to 20 pieces/plate andco-cultured for 2 days at 25° C. under 2000 lux condition. After theco-culturing, the pieces were cultured under sterile conditions for 7days in MS medium (pH 5.8) comprising 3% sucrose, 0.5 ppm BAP, 0.1 ppmNAA, 250 μg/ml carbenicillin and 0.8% agar.

(3) Selection and Culturing of Transformants

After sterile culture, the pieces were transferred to MS medium (pH 5.8)comprising 3% sucrose, 0.5 ppm BAP, 0.1 ppm NAA, 250 μg/mlcarbenicillin, 50 μg/ml kanamycin and 0.8% agar and cultured at 25° C.under 2000 lux condition. The pieces were sub-cultured about every 2weeks, and regeneration was observed after about 2 to 3 months fromthose plants that were inoculated with Agrobacterium.

The re-differentiated individuals were transferred to MS medium (pH 5.8)comprising 3% sucrose, 0.3 ppm BAP, 500 μg/ml carbenicillin and 0.8%agar, and were sub-cultured about every 2 weeks. When there-differentiated individuals reached a size of about 3 cm, the cuttingof them were inserted and planted into ½-fold MS agar medium comprising500 μg/ml of carbenicillin and allowed to take root.

(4) Lettuce Acclimation and Seed Sampling

In the state in which the re-differentiated individuals took root andthe shoots had grown to 1 to 2 cm, the shoots were cut, and the cuttingswere planted and allowed to take root in vermiculite immersed in a500-fold diluted aqueous solution of Hyponex. The plants were acclimatedby gradually opening the cover of the plant box to provide ventilation.After the plants had been sufficiently acclimated, they were permanentlyplanted in Polypot comprising Kureha Horticultural Soil (Kureha engeibaido) in a closed greenhouse (maximum air temperature: 30° C., naturalphotoperiod). Then, they were allowed to be bolting and flowering, andseeds were sampled.

EXAMPLE 3 Cloning of LBVV RNA2 Gene

Contaminated soil was sampled from a lettuce (cultivar: Cisco) field inKagawa Prefecture, Japan that exhibited characteristic big-vein symptomsin 1997. The virus was maintained in resting spores in dry soil kept inthe laboratory. Cisco, a cultivar of lettuce, was used for viruspurification, and the virus was inoculated by regular transfer in soil.

Virus purification and RNA purification were carried out in accordancewith Example 1. Synthesis of cDNA and determination of nucleotidesequence were carried out in accordance with the method of C. F. Fazeli& M. A. Rezaian (Journal of General Virology, 81, 605-615) using agenome walking method, in which the sequence is extended by synthesizingprimer to the downstream direction. First, virus-specific 5LB5R3 primer(AGCTCTGAACAACGACATG/SEQ ID NO: 16) was produced based on Example 1, anda first cDNA was synthesized with SUPERSCRIPT™ II RNase H⁻ ReverseTranscriptase using an RNA from the purified LBVV as a template. Next, asecond cDNA was synthesized with Klenow Fragment (Takara) usinguniversal primer dN6 (5′-GCCGGAGCTCTGCAGAATTCNNNNNN-3′/SEQ ID NO: 14).After removing excess primer with the GlassMax DNA Isolation SpinCartridge System (Gibco BRL), PCR was carried out using thevirus-specific primer and universal primer(5′-GCCGGAGCTCTGCAGAATTC-3′/SEQ ID NO: 15) and the resulting PCR productwas cloned using pGEM®-T Easy Vector Systems. Then, the nucleotidesequence was determined. The same procedure was then repeated four timesto determine up to 5177 nucleotides. Determination of the 3′-terminus ofRNA2 was carried out by 5′RACE (Note: since purified LBVV RNA containsboth a positive-strand and a negative-strand, not only the 5′-terminusbut also 3′-terminus can be determined by 5′RACE), and a 6078nucleotides sequence was determined that comprised genes for fiveproteins encoded by LBVV (SEQ ID NO: 1). Furthermore, the 500- to 700-bpPCR products from RNA2 were recloned using mutually overlapping virusspecific primers. At least three clones were sequenced from each region,and the nucleotide sequence of RNA2 was confirmed.

A 6078 nucleotides sequence was determined using the above method. Thisgene encoded five proteins. Protein 1 (coat protein: Example 1) had atranslation initiation site at nucleotide 209 and encoded 397 aminoacids (SEQ ID NO: 2), protein 2 had a translation initiation site atnucleotide 1492 and encoded 333 amino acids (SEQ ID NO: 3), protein 3had a translation initiation site at nucleotide 2616 and encoded 290amino acids (SEQ ID NO: 4), protein 4 had a translation initiation siteat nucleotide 3842 and encoded 164 amino acids (SEQ ID NO: 5), andprotein 5 had a translation initiation site at nucleotide 4529 andencoded 368 amino acids (SEQ ID NO: 6). When the homology of the aminoacid sequences was compared with other viruses, only protein 1 (coatprotein) was observed to be homologous to the nucleocapsid protein (coatprotein) of viruses belonging to the family Rhabdoviridae.

EXAMPLE 4 Cloning of LBVV Polymerase Gene

Contaminated soil was sampled from a lettuce (cultivar: Cisco) field inKagawa Prefecture, Japan that exhibited characteristic big-vein symptomsin 1997. The virus was maintained in resting spores in dry soil kept inthe laboratory. Cisco, a cultivar of lettuce, was used for viruspurification, and the virus was inoculated by regular transfer in soil.

Virus purification was carried out in the same manner as the procedurefor virus purification of Example 1. Extraction of highly pure LBVV RNAwas carried out in the manner described below. After treatment of thepurified virus with Proteinase K-SDS, it was extracted withphenol/chloroform and precipitated with ethanol. Next, after the viralnucleic acid was treated with DNase and further purified with The RNaid®Kit (BIO 101), a 7.3 kb RNA of two LBVV RNAs was isolated by 1% agarosegel (SeaPlaque GTG agarose, FMC) electrophoresis and used for synthesisof cDNA. Synthesis of cDNA was carried out in accordance with the methodof P. Froussard (Nucleic Acids Research, 20, 2900). In brief, a firstcDNA was synthesized with the SUPERSCRIPT™ II RNase H⁻ ReverseTranscriptase using universal primer-dN6(5′-GCCGGAGCTCTGCAGAATTCNNNNNN-3′/SEQ ID NO: 14). Next, a second cDNAwas synthesized with Klenow Fragment, PCR was carried out usinguniversal primer (5′-GCCGGAGCTCTGCAGAATTC-3′/SEQ ID NO: 15), and theresulting PCR product was cloned using pGEM®-T Easy Vector Systems Then,the nucleotide sequence was determined.

Eight partial LBVV polymerase gene fragments of about 500 bp wereobtained. Both terminals of the polymerase gene were filled by 5′RACEand the gaps between the fragments were filled in by RT-PCR, therebydetermining a 6793 nucleotides sequence that contained the full-lengthpolymerase gene (SEQ ID NO: 12). Furthermore, the 500- to 700-bp PCRproducts from polymerase gene were recloned using mutually overlappingvirus-specific primers. At least three clones were sequenced from eachregion, and the nucleotide sequence of polymerase gene was confirmed.

This gene encoded 2040 amino acids with a translation initiation site atnucleotide 337 (SEQ ID NO: 13). When the homology of the amino acidsequence was compared with other viruses, it was confirmed that theprotein is homologous to the polymerases of viruses belonging to thefamily Mononegavirales, especially, viruses belonging to the familyRhabdoviridae, and retained four motifs considered to be responsible forpolymerase activity.

INDUSTRIAL APPLICABILITY

According to the present invention, nucleic acids encoding proteins ofLBVV were isolated, and their primary structure was elucidated. Ittherefore became possible to produce a lettuce plant having resistanceto the virus by expressing the nucleic acid or its antisense nucleicacid in lettuce. In addition, it became possible to make a diagnosis ofinfection with LBVV by detecting the nucleic acid or protein encodedthereby.

1. An isolated nucleic acid encoding a coat protein of lettuce big-veinvirus, said nucleic acid selected from the group consisting of: (a) anucleic acid encoding a protein comprising the amino acid sequence ofSEQ ID NO: 2; and (b) the nucleic acid of (a) comprising bases 209-1400of the nucleotide sequence of SEQ ID NO:1.
 2. The nucleic acid accordingto claim 1, wherein the nucleic acid is an RNA.
 3. The nucleic acidaccording to claim 1, wherein the nucleic acid is a DNA.
 4. An isolatedDNA encoding an RNA that suppresses the production of a lettuce big-veinvirus protein comprising the amino acid sequence of SEQ ID NO: 2,wherein the DNA is any one of the following (a) to (d): (a) a DNAencoding a sense RNA which has a complementarity of 90% or more to RNAthat is completely complementary to the RNA according to claim 2; (b) aDNA encoding an antisense RNA which has a complementarity of 90% or moreto the RNA according to claim 2; (c) a DNA encoding the RNA according toclaim 2; and (d) a DNA encoding an antisense RNA which is completelycomplementary to the RNA according to claim
 2. 5. A vector comprisingthe nucleic acid according to claim
 3. 6. A transformed cell comprisingthe nucleic acid according to claim
 3. 7. A method of producing aprotein, wherein said method comprises the steps of: (a) culturing thetransformed cell of claim 6; and (b) recovering an expressed proteinfrom said transformed cell or its culture supernatant.
 8. A vectorcomprising the DNA according to claim
 4. 9. A transformed lettuce cellthat comprises the nucleic acid according to claim
 1. 10. A transformedlettuce cell that comprises the DNA according to claim
 4. 11. Atransformed lettuce cell that comprises the vector according to claim 5or claim
 8. 12. A transformed lettuce plant comprising the transformedlettuce cell according to claim 9 or claim
 10. 13. A transformed lettuceplant comprising the transformed lettuce cell according to claim
 11. 14.A transformed lettuce plant that is a progeny or a clone of thetransformed lettuce plant according to claim
 12. 15. A transformedlettuce plant that is a progeny or a clone of the transformed lettuceplant according to claim
 13. 16. A propagation material of thetransformed lettuce plant according to claim 12, wherein the propagationmaterial comprises said nucleic acid.
 17. A propagation material of thetransformed lettuce plant according to claim 13, wherein the propagationmaterial comprises said nucleic acid.
 18. A propagation material of thetransformed lettuce plant according to claim 14, wherein the propagationmaterial comprises said nucleic acid.
 19. A propagation material of thetransformed lettuce plant according to claim 15, wherein the propagationmaterial comprises said nucleic acid.
 20. A transformed cell comprisingthe vector according to claim
 5. 21. A method of producing a protein,wherein said method comprises the steps of: (a) culturing thetransformed cell of claim 20; and (b) recovering an expressed proteinfrom said transformed cell or its culture supernatant.